Protective Effect of Caffeic Acid Phenethyl Ester (CAPE) on Amiodarone-Induced Pulmonary Fibrosisin Rat.

Treatment with amiodarone, a commonly prescribed antidysrhythmic agent, is associated with pulmonary fibrosis (PF) which is a commonly progressive and untreatable disease. Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant and an active anti-inflammatory , anticancer, antimicrobial and antioxidant component of propolis (bee glue; a resinous hive product collected by honey bees). In the current study, the effects of CAPE on amiodarone-induced pulmonary fibrosis in rat were investigated. Male rats were divided in to 4 groups. The first group only received amiodarone (6.25 mg/Kg) on first and third day. The second group received only vehicle (distilled water) with the same volume and in the same time as the first group. The third and fourth groups received amiodarone and were treated with CAPE , 5 and 10 µmol /day respectively, from 2 days before the first dose of amiodarone and until 21 days after the second dose of amiodarone. At the end of treatment course, lung tissue was removed for histopathology and biochemical evaluations. Malondialdehyde (MDA) concentration, myeloperoxidase MPO) and super oxide dismutase (SOD) activities were determined in lung tissue. Histopathological evaluation was performed using light microscopy. MDA level and the activity of myeloperoxidase and superoxide dismutase enzymes significantly decreased in the group which was treated with CAPE (5 µmol/Kg). However, 10 µmol/Kg CAPE had not such an effect. Both doses of CAPE could histopathologically reduce the fibrogenic effects of amiodarone . CAPE was shown to be effective in reducing amiodarone-induced pulmonary fibrosis with the dose of 5 µmol/Kg.


Introduction
and essentially untreatable disease with a fatal outcome, which is characterized by an altered cellular composition of the alveolar region with excessive deposition of collagen. Typical features in this disease include dyspnea, diffuse affected by this disease. In the United States there are over 200,000 patients with pulmonary remains incompletely understood, but lung disorders (1-3).
It has been suggested in the last decades that the main responsible agent in PF is reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, peroxynitrites and hydroxyl radicals, which are generated also in normal physiological conditions in the human body. These ROS are capable of initiating and promoting oxidative The antioxidant serves as a defensive factor against free radicals in the body. Enzymes such as superoxide dismutase, catalase and glutathione peroxidase are the main system that opposes oxidation and act as a protective mechanism. If the free radicals production becomes more than the capacity of enzymatic system, the second line of defense (vitamins) may come to action. Antioxidant such as vitamins C and E quench free radicals and become oxidized and inactive (3)(4)(5).
Treatment with amiodarone (AM), commonly prescribed as an antiarhythmic agent, may decrease the incidence of mortality following myocardial infarction or due to out-of-hospital sudden cardiac arrest. However amiodarone administration is associated with few adverse effects including pulmonary injury resulting for amiodarone pulmonary injury include direct toxicity to lung tissue, hypersensitivity reaction to amiodarone, and enhanced oxidative stress. Alteration of membrane properties and activation of alveolar macrophages and cytokine release are the other proposed mechanisms of amiodarone toxicity (6, 7).
Propolis or bee glue is a resinous hive product collected by honey bees from plant exudates (especially conifer buds) and contains more than 160 constituents. It is used to repair hive cracks by bees. Historically it has been used for various purposes, especially as a medicine and has been used empirically as a traditional remedy in folk medicine for centuries. Recently, propolis has been extensively marketed by the pharmaceutical industries as an alternative medicine and as the health-food in various parts of the world. Propolis has been claimed to improve the health and prevent diseases such as diabetes, heart diseases, and even cancer. Flavonoids are thought to be responsible for many of its biological and pharmacological activities including anticancer, effects (7-9). Several investigations on propolis in Eastern Europe and South America have are powerful antioxidants which are capable to scavenge free radicals (10).
Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant and is an active antiwas shown to exert its antioxidant activity by suppressing lipid peroxidation, scavenging the reactive oxygen species, and inhibiting xanthine oxidase and nitric oxide synthase activities. It may reduce the activity of superoxide dismutase (11). It was also found to inhibit 5-lipoxygenase catalysed oxygenation of linoleic acid and arachidonic through a glucocorticoid receptor independent mechanism (12) .
In the current study we investigated the of CAPE on amiodarone-induced pulmonary effects include: myeloperoxidase (MPO) activity, malondialdehide (MDA) concentration (indicators of oxidative stress), superoxide dismutase (SOD) activity (an antioxidant enzyme), and histopathological examination (12).

Animals
Male Wistar albino rats weighing 180-200 g were purchased from Animal house and research center, Jundishapur University of Medical Sciences, Ahwaz, Iran. The animals were kept on a standard food-pellet and tap-water ad libitum. The rats were housed in polycarbonate cages (5 animals per cage) and kept in an air-conditioned a relative humidity of 50 ± 5%. The animals' room was on a 12 h light-dark period cycle. amiodarone

Chemical agents
Amiodarone was dissolved in distilled water at 60 C and allowed to be cooled in room temperature before instillation (13). The rats were anesthetized with sodium methohexital (40 mg/Kg). Two doses of amiodarone (6.25 mg/Kg) were given what is designated as day 1 and the second on day 3 (14).

Drugs treatment groups
The animals divided into the following groups (10 rats per group (n = 10)): Group 1: This group received only intra-tracheal amiodarone as positive control. Group 2: The rats in this group received only intra-tracheal vehicle (distilled water) as negative control. of amiodarone instillation until 3 weeks after the second dose of amiodarone instillation.
instillation until 3 weeks after the second dose of amiodarone (15). The vehicle in all solutions was distilled water.

Collecting tissue samples
After the last injection of CAPE, rats were killed by sternotomy, following deep etherinduced anesthesia. After mid-line sternotomy, both lungs were removed. Right lungs were preserved in formaldehyde solution (10%) for histopathological evaluation and left lungs were divided into 3 parts, washed with heparinized examinations.
Biochemical assays a) Malondialdehyde (MDA) assay: This assay is based on the reaction of MDA with thiobarbituric acid (TBA); forming a MDA-TBA2 adduct that absorbs at 532 nm (16,17). The assay was performed through the above mentioned kit and via the manufacturer instructions.
b) Myeloperoxidase (MPO) assay: In this assay, H 2 O 2 is broken down by MPO released from samples by homogenization in detergent. The produced radical oxygen (O) combines with a hydrogen-donating chromogen which is thereby converted to a colored compound. The appearance of this colored compound is measured, over time, with a spectrophotometer (at 450 nm) to determine MPO activity in a sample (18). The assay was performed through the above mentioned kit and via the manufacturer protocol. c) Superoxide dismutase (SOD) assay: The assay's method is based on the SOD-mediated increase in the rate of autoxidation of 5, 6, 6a, 11btetrahydro-3, 9, 10-trihydroxybenzo [c] chromophore with maximum absorbance at 525 nm. The chromophore has not been isolated or characterized (19). The assay is also performed through the above mentioned kit and via the manufacturer protocol.

Histopathological examination and scoring
counterstained with hematoxylin and eosin (H&E) (20). The slides were examined by light microscopy and photographed (20).According to the literature, the following scoring codes were used: with thickening of alveolar or bronchial wall; 2 = Moderate thickening of bronchial wall with mild damage to lung architecture; 3 = Severe distortion of lung structure and presence of

Statistical analysis Statistical comparison was made by one-way
Tukey's test. Data are presented as mean ± SEM.

MDA concentrations in lung tissue
) reduce the MDA concentration in amiodaronetreated group. However, higher concentration amiodarone-treated lung tissue (Figure 1).

MPO activity in lung tissue
reduce the MPO activity in amiodarone-treated group. However, higher concentration of amiodarone-treated lung tissue (Figure 2).

SOD activity in lung tissue
reduce the SOD activity in amiodarone-treated group. However, higher concentration of CAPE amiodarone-treated lung tissue (Figure 3).

Light microscopic results
Histopathological evaluation of the pulmonary tissue was performed with light microscopy for the 4 different experimental rats in negative control group, which received distilled water, showed normal lung structure and no lesion was obvious (Figure 4). Three weeks after the second amiodarone instillation, associated with perivascular and peribronchial groups, considerable improvement in tissue structure was observed. In these groups, less thickening was evident (Figures 6 and 7).

Discussion
characterized by an accumulation of alveolar macrophages and neutrophils in the lower respiratory tract, parenchymal cell injury, and   The mechanisms proposed for amiodaronerelease; (ii) cell membrane perturbation; (iii) phospholipidosis promotion; (iv) altered Ca 2+ cellular homeostasis; (v) mitochondrial dysfunction; and (vi) free radical production. Although the exact mechanism is not fully understand (24). In this model of amiodarone-induced lung injury we observed a decrease in malondialdehyde concentrations, and myeloperoxidase and superoxide dismutase activities in lung in histopathological examination there were structural changes in amiodarone-treated group in comparison to negative control group (D/W treated animals). A pronounced decreased in groups. Previous researches showed free radical anti-endotoxine activities for CAPE (12,21,25).
in the amiodarone treated group in comparison with the vehicle treated rats, and CAPE with the even less than the negative control group .As MDA is a highly reactive agent produced as a byproduct of polyunsaturated fatty acid peroxidation and arachidonic acid metabolism, (26) we can say that low-dose CAPE is a very potent anti-lipid peroxidation and antioxidant agent. In contrast to previous studies(12, 21), MDA concentration in comparison with negative control and low dose CAPE groups. decrease in MPO activity in the low-dose CAPE treated animals in comparison to positive control. When accumulated and activated, neutrophils can release myeloperoxidase, an 115,000-dalton protein which can interact with H 2 0 2 , a product of both mononuclear phagocytes and neutrophils, to form a highly toxic anion, which can initiate oxidative stress (12, 22). Our results suggest that low dose CAPE could successfully lung toxicity. However; high dose of CAPE had opposite effect compared to the previous studies; activity in comparison with negative control and low dose CAPE groups (12, 21).
is that administration of high dose of CAPE (10 to another species of active oxygen. It should evaluate more, as the other studies do not show such an effect. Also it may be an observer error   or a technical defect in some studies. activity in positive control group in comparison with negative control. It is reasonable, as in the normal airway mucosa, antioxidant defense systems, such as superoxide dismutase (SOD), glutathione peroxidase, and catalase exist to protect the mucosa from various oxidant stimuli and in fact this enzyme has applied for the treatment of PF in some studies (27,28) and they showed inhibitory effects of SOD on kg CAPE decreased the need for compensatory increase in SOD activity against amiodaroneinduced oxidative injury.
In this study, we concluded that systemic amiodarone-induced lung injury successfully. Therefore; CAPE may be a potential treatment investigations are required to elucidate the exact mechanism of this novel agent in pulmonary